Radiolabelled fluorobenzamide analogues, their synthesis and use in diagnostic imaging

ABSTRACT

Fluoroalkoxybenzamide compounds which selectively bind Sigma-2 receptors are disclosed. These compounds, when labelled with  18 F, can be used as radiotracers for imaging of tumors by positron emission tomography (PET). In addition, these compounds, when labelled with  123 I, can be used as radiotracers for imaging of tumors by single photon emission computed tomography (SPECT). Methods for synthesis of these compounds are also disclosed.

RELATED APPLICATIONS

The present application is a division of U.S. patent application Ser.No. 12/643,175, filed Dec. 21, 2009, which is a division of U.S. patentapplication Ser. No. 11/757,246, filed Jun. 1, 2007, now patented, whichis a continuation-in-part of U.S. patent application Ser. No. 10/903,771filed Jul. 30, 2004, now patented, which claims priority to U.S.Provisional Application 60/491,582 filed Jul. 31, 2003. Theseapplications are hereby incorporated by reference, each in its entirety.

INTRODUCTION

Sigma receptors are a class of receptors that are expressed in manynormal tissues, including liver, kidneys, endocrine glands, and thecentral nervous system (CNS) (Walker, J. M., et al. Pharmacol Rev 42:355-402 1990). It has been well established that there are at least twotypes of sigma receptors, sigma-1 (σ₁) and sigma-2 (σ₂) (Walker, J. M.,et al. Pharmacol Rev 42, 355-402, 1990). Overexpression of σ₂ receptorshas been reported in a variety of human and murine tumors (Bem, W. T.,et al., Cancer Res 51: 6558-6562, 1991; Vilner, B. J., et al., In:Multiple sigma and PCP receptor ligands: mechanisms for neuromodulationand neuroprotection?, Kamenka, J. M., and Domino, E. F., ed, Ann Arbor(Mich), 7 NPP Books, p. 341-353, 1992; Mach, R. H., et al., Cancer Res.57: 156-161, 1997).

Searches for σ₂ selective ligands has led to the identification of anumber compounds having modest to high selectivity for σ₂ versus σ₁receptors (FIG. 5). These include CB-184 (10), CB-64D (11), BIMU-1 (12)(Bowen, W. D., et al., Eur. J. Pharmacol. 278: 257-260, 1995; Bonhaus,D. W., et al., J. Pharmacol. Exp. Ther. 267: 96, 1993) and PB-167 (13)(Colabufo, N. A., et al., J. Pharmacy and Pharmacology 57: 1453-1459,2005; Kassiou, M., et al., Bioorganic and Medicinal Chemistry, 13:3623-3626, 2005; Berardi, F., et al., J. Med. Chem. 2004, 47: 2308-2317)as well as certain benzamide analogs (14-16) (Mach, R. H., et al.,Bioorg. Med. Chem. 11: 225, 2003; Huang, V., et al., J. Med. Chem. 44:1815, 2001; U.S. patent application Ser. No. 10/903,771 to Mach et al).We previously reported the evaluation of several ¹¹C, ⁷⁶Br and^(125/123)I radiolabelled conformationally-flexible benzamide analogsusing EMT-6 tumor-bearing female Balb/c mice (Tu, Z., et al., Nucl. Med.Biol. 32: 423-430, 2005; Xu, J., et al., Eur. J. Pharmacol. 21: 525(1-3): 8-17, 2005; Hou, C., et al., Nucl. Med. Biol. February, 33:203-9, 2006). Initial in vivo studies of5-methyl-2-[¹¹C]-methoxy-N-[2-(6,7-dimethoxy-3,4-dihydro-1Hisoquinolin-2-yl)-butyl]-benzamide and5-[⁷⁶Br]-bromo-2,3-dimethoxy-N-[2-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-butyl]-benzamideindicated that these compounds were potential radiopharmaceuticals forimaging solid tumors and their proliferative status with positronemission tomography (PET). However, the radionuclide properties of ⁷⁶Brand ¹¹C make these isotopes less than ideal for PET imaging. Forexample, images produced by PET using ⁷⁶Br as a radiotracer are oftenblurry, (Laforest. R., et al., IEEE Transactions on Nuclear Science, 49:2119-2126, 2002), and the short half-life of ¹¹C (t_(1/2)=20.4 min)places time constraints on tracer synthesis and duration of scansessions. Contrast between tumor and normal tissues can be less thansatisfactory when a σ₂-selective radiotracer tagged with ¹¹C is used inPET imaging. Accordingly, alternative σ₂-selective ligands for use asradiotracers in PET imaging are needed.

SUMMARY

The present inventors have developed a series of compounds which can beused as radiolabels for diagnostic imaging, in particular positronemission tomography (PET) imaging of tumors. The compounds selectivelybind Sigma receptors, and in particular bind Sigma-2 receptors inpreference to Sigma-1 receptors. The compounds also selectively bind totumor cells, and thus can also be used as tracers for detecting tumorcells. In addition, because in some embodiments, the compounds comprisethe radioisotope ¹⁸F, they can be used as radiotracers for imagingtumors using PET.

In some embodiments, a tracer the present teachings is afluoroalkoxybenzamide compound having a structure

wherein m is an integer from 1 to about 10, n is an integer from 1 toabout 10, and R₁ and R₂, are each independently selected from the groupconsisting of H, a halogen selected from the group consisting of I, Br,Cl and F, a C₁₋₄ alkoxy, a C₁₋₄ alkyl, a C₁₋₄ fluoroalkyl, a C₁fluoroalkoxy, CF₃, OCF₃, SCH₃, SCF₃, and NH₂, or a salt thereof.

In some embodiments, a compound of the present teachings can include atleast one ¹⁸F isotope. A compound of these embodiments can be aradiolabelled fluoroalkoxybenzamide compound having a structure

wherein in is an integer from 1 to about 10, n is an integer from 1 toabout 10, and R₁ and R₂ are each independently selected from the groupconsisting of H, a halogen selected from the group consisting of I, Br,Cl and F, a C₁₋₄ alkoxy, a C₁₋₄ alkyl, a C₁₋₄ fluoroalkyl, a C₁₋₄fluoroalkoxy, CF₃, OCF₃, SCH₃, SCF₃, and NH₂, or a salt thereof.

Other embodiments of the present teachings include methods ofsynthesizing fluoroalkoxybenzamide compound of structure

These methods comprise reacting a compound of structure

with an fluorinated compound such as

wherein m is an integer from 1 to about 10, n is an integer from 1 toabout 10, and R₁ and R₂ are each independently selected from the groupconsisting of H, a halogen selected from the group consisting of Br, Cland F, a C₁₋₄ alkoxy, a C₁₋₄ alkyl, a C₁₋₄ fluoroalkyl, a C₁₋₄fluoroalkoxy, CF₃, OCF₃, SCH₃, SCF₃, and NH₂. In some aspects of theseembodiments, m can be 2, n can be 2, R₁ can be H, and R₂ can be CH₃. Insome aspects, these methods can further comprise reacting

thereby forming

In other aspects, m can be 2, n can be 4, R₁ can be selected from thecroup consisting of OCH₃ and H, and R₂ can be selected from the groupconsisting of Br, CH₃, and I. In yet other aspects, m=2, n=4, R₁ is H,and R₂ is I.

In yet other embodiments of the present teachings, the inventorsdisclose methods for synthesizing radiolabelled fluoroalkoxybenzamidecompounds of structure

These methods comprise forming a mixture comprising i) an organicsolvent, ii) a compound of structure

iii) ¹⁸F, iv)4,7,13,16,21,24-Hexaoxa-1,10-diazabicyclo[8.8.8]-hexacosane and v) apotassium salt, wherein in is an integer from 1 to about 10, n is aninteger from 1 to about 10, R₁ and R₂, are each independently selectedfrom the group consisting of H, a halogen selected from the groupconsisting of Br, Cl and F, a C₁₋₄ alkoxy, a C₁₋₄ alkyl, a C₁₋₄fluoroalkyl, a C₁₋₄ fluoroalkoxy, CF₃, OCF₃, SCH₃, SCF₃, and NH₂. Insome aspects of these methods, in can be 2, n can be 4, R₁ can beselected from the group consisting of H and OCH₃, and R₂ can be selectedfrom the group consisting of CH₃, Br and I. In yet other aspects ofthese methods, m=2, n=4, R₁ is OCH₃, and R₂ is I. In addition, invarious aspects, the potassium salt can be K₂CO₃, and the organicsolvent can be dimethyl sulfoxide, acetonitrile or a combinationthereof. Furthermore, in various aspects; the methods can includeheating a mixture.

In additional embodiments of the present teachings, the inventorsdisclose methods of imaging a tumor in a mammal such as a human. Thesemethods comprise administering to the mammal a radiolabelledfluoroalkoxybenzamide compound of structure.

wherein m is an integer from 1 to about 10, n is an integer from 1 toabout 10, and R₁ and R₂ are each independently selected from the groupconsisting of H, a halogen selected from the group consisting of I, Br,Cl and F, a C₁₋₄ alkoxy, a C₁₋₄ alkyl, a C₁₋₄ fluoroalkyl, a C₁₋₄fluoroalkoxy, CF₃, OCF₃, SCH₃, SCF₃, and NH₂, or a salt thereof; andsubjecting the mammal to positron emission tomography (PET) scanning. Insome aspects, m can be 2, n can be 4, R₁ can be selected from the groupconsisting of H and OCH₃, and R₂ can be selected from the groupconsisting of CH₃, Br and I.

In various aspects of the above embodiments, a fluoroalkoxybenzamidecompound or a salt thereof can include particular molecular species,such as,

or salts thereof.

In various aspects of these embodiments, a radiolabelledfluoroalkoxybenzamide compound or a salt thereof can include particularmolecular species, such as,

or salts thereof.

In additional embodiments of the present teachings, the inventorsdisclose methods for synthesizing compounds of structure

wherein m is an integer from 1 to about 10, n is an integer from 1 toabout 10, and R is H or a C₁₋₄ alkoxy such as a methoxy. These methodscomprise:

stannylating a compound of structure

to form a compound of structure

and iodinating a compound of structure

In some configurations, the stannylated compound can be formed bystannylating a compound of structure

In some aspects of these methods, m can be 2 and n can be 4.

In yet other embodiments oldie present teachings, the inventors discloseiodine-123 radiolabelled fluoroalkoxybenzamide compounds of structure

wherein in is an integer from 1 to about 10, n is an integer from 1 toabout 10, and R can be H, a halogen selected from the group consistingof I, Br, Cl and F, a C₁₋₄ alkoxy, a C₁₋₄ alkyl, a C₁₋₄ fluoroalkyl, aC₁₋₄ fluoroalkoxy, CF₃, OCF₃, SCH₃, SCF₃, and NH₂, and salts thereof. Invarious configurations, in can be 2, n can be 4, R can be H or aC₁₋₄alkoxy such as a methoxy.

In related embodiments, the inventors disclose methods for synthesizingthese radioiodinated compounds. In various configurations, these methodsinclude reacting a compound of structure

with [¹²³I]NaI and an oxidant, wherein m is an integer from 1 to about10, n is an integer from 1 to about 10, A₁, A₂ and A₃ are eachindependently a C₁₋₄ alkyl, and R is selected from the group consistingof H, a halogen selected from the group consisting oil, Br, Cl and F, aC₁₋₄ alkoxy, a C₁₋₄ alkyl, a C₁₋₄ fluoroalkyl, a fluoroalkoxy, CF₃,OCF₃, SCH₃, SCF₃, and NH₂. In various aspects, R can be H or a C₁₋₄alkoxy such as a methoxy; A₁, A₂ and A₃ can each be independently abutyl moiety selected from an n-butyl moiety, an iso-butyl moiety, asec-butyl moiety, and a tert-butyl moiety. In some configurations, A₁,A₂ and A₃ can each be an n-butyl moiety, m can be 2 and n can be 4. Inaddition, in various aspects, the oxidant can be peracetic acid,hydrogen peroxide, chloramine T (N-chloro-p-toluenesulfonamide sodiumsalt) or a combination thereof.

In some aspects of these embodiments, the methods can further includestannylating a compound of structure

to form a compound of structure

In yet other embodiments, the present teachings include methods ofimaging a solid tumor in a mammal such as a human. In variousconfigurations, these methods include administering to the mammal aradioiodinated compound described above, and subjecting the mammal tosingle photon emission computed tomouraphy (SPECT) imaging.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates scheme I for synthesis of some compounds of thepresent teachings.

FIG. 2 illustrates scheme II for synthesis of some compounds of thepresent teachings.

FIG. 3 illustrates scheme III for synthesis of some compounds of thepresent teachings.

FIG. 4 illustrates scheme IV for synthesis of some compounds of thepresent teachings.

FIG. 5 illustrates structure and properties of several σ₂ selectiveligands.

FIG. 6 illustrates tumor:organ ratios for the ¹⁸F-labeled σ₂ selectiveligands, 3c-f, at 1 h (top) and 2 h (bottom) after i.v. injection intofemale Balb/c mice bearing EMT-6 tumors.

FIG. 7 presents a comparison of the tumor:fat and tumor:muscle ratiosfor [¹⁸F]3c or [¹⁸F]3f when there is no-carrier-added and when the σ₁and σ₂ receptors are blocked with 1 mg/kg of YUN-143. All values wereobtained 1 h after injection of the radiotracer.

FIG. 8 illustrates microPET and microCT images of EMT-6 tumors in femaleBalb/c mice. All MicroPET images were acquired 1 h after i.v. injectionof either [¹⁸F]3c or [¹⁸F]3f.

FIG. 9 illustrates an image of a glioma using [¹⁸F]3f of the presentteachings compared to [¹⁸F]FDG.

DETAILED DESCRIPTION

The present inventors have developed a series of compounds which can beused as radiolabels for diagnostic imaging, in particular positronemission tomography (PET) imaging of tumors. The compounds selectivelybind Sigma receptors, and in particular bind Sigma-2 receptors inpreference to Sigma-1 receptors. The compounds also selectively bind totumor cells, and thus can be used as tracers for detecting tumor cells.Without being limited by theory, it is generally believed that manytypes of tumor cells have a high density of sigma-2 receptors, andtherefore compounds of the present teachings are effective tracers fordetecting tumors by virtue of the compounds' affinity for the sigma-2receptors. In addition, because in some embodiments, the compoundscomprise the radioisotope ¹⁸F, a preferred isotope for imaging bypositron emission tomography (PET) they are effective as radiotracersfor PET imaging of tumors in humans or other mammals. Furthermore, insome embodiments, the compounds comprise the radioisotope ¹²³I, apreferred isotope for imaging by single photon emission computedtomography (SPECT). These compounds are effective as radiotracers forSPECT imaging of tumors in humans or other mammals.

The present inventors have synthesized several novel conformationallyflexible benzamide analogues having a moderate to high binding affinityand selectivity for σ₂ receptors (Table I). Four of these compounds wereselected as candidates for developing ¹⁸F-labeled PET probes to imagethe σ₂ receptor status of solid tumors. [¹⁸F]3c, [¹⁸F]3d, [¹⁸F]3e, and[¹⁸F]3f, were successfully synthesized and evaluated as potentialradiotracers for imaging EMT-6 tumors in female Balb/c mice. Of the four¹⁸F-labeled analogues, [¹⁸F]3c and [¹⁸F]3f had the best biodistributionkinetics and tumor:normal tissue ratios. Blocking studies confirmed thatthe uptake of [¹⁸F]3c and [¹⁸F]3f was σ₂-receptor mediated. Our studiesindicate that various compounds of the present teachings, including[¹⁸F]3c and [¹⁸F]3f, are acceptable agents for detecting and imagingsolid tumors and their σ₂ receptor status with PET.

The present inventors devised a design strategy for generatingσ₂-selective ligands of the present teachings. This strategy involvedreplacing the ortho methoxy group of ¹¹C-labelled benzamide analogs (Xu,J., et al., Eur. J. Pharmacol. 21; 525 (1-3): 8-17, 2005) with a2-fluoroethyl group as shown in Scheme I (FIG. 1) and in Examples below.

The following examples are illustrative of the various embodiments ofthe present teachings. The examples are not intended to limit the scopeof the claims. The methods described herein utilize laboratorytechniques well known to skilled artisans, and guidance can be found inlaboratory manuals and textbooks such as Sambrook, J., et al., MolecularCloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 2001; Spector, D. L. et al., Cells: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1998; and Harlow, E., Using Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1999; Hedrickson et al., Organic Chemistry 3rd edition, McGraw Hill, NewYork, 1970; Carruthers, W., and Coldham, I., Modern Methods of OrganicSynthesis (4th Edition), Cambridge University Press, Cambridge, U.K.,2004; Curati, W. L., Imaging in Oncology, Cambridge University Press,Cambridge, U.K., 1998; Welch, M. J., and Redvanly, C. S., eds. Handbookof Radiopharmaceuticals: Radiochemistry and Applications, J. Wiley, NewYork, 2003.

In the experiments described in herein, all reagents were purchased fromcommercial suppliers and used without further purification unlessotherwise stated. Tetrahydrofuran (THF) was distilled from sodiumhydride immediately prior to use. Anhydrous toluene was distilled fromsodium/toluene shortly before use. All anhydrous reactions were carriedout in oven-dried glassware under an inert nitrogen atmosphere unlessotherwise stated. When the reactions involved extraction withdichloromethane (CH₂Cl₂), chloroform (CHCl₃), ethyl acetate (EtOAc), orethyl ether (Et₂O), the organic solutions were dried with anhydrousNa₂SO₄ and concentrated with a rotary evaporator under reduced pressure.Flash column chromatography was conducted using silica gel 60a, “40Micron Flash” [32-63 μm] (Scientific Adsorbents, Inc.). Melting pointswere determined using the MEL-TEMP 3.0 apparatus and left uncorrected.¹H NMR spectra were recorded at 300 MHz on a Varian Mercury-VXspectrometer with CDCl₃ as solvent and tetramethylsilane (TMS) as theinternal standard. All chemical shift values are reported in ppm ( )Elemental analyses (C, H, N) were determined by Atlantic Microlab, Inc.

EXAMPLE 1

This example demonstrates reactions yielding fluoroalkoxy2-hydroxybenzamide analogs. As illustrated in FIG. 1, Scheme I involvescondensation of compounds 1a and 1b with a substituted salicylic acid togive the corresponding substituted 2-hydroxybenzamide analogs, 2a-e.Alkylation of the ortho hydroxyl group with 2-bromo-1-fluoroethane usingpotassium carbonate as a base produced 3a-c in moderate to high yield.Compound 3f was prepared by iodination of the corresponding tinprecursor, 3g, which was prepared from 3b using standard stannylationreaction conditions. Compounds, 3a-f, were then converted into eitherthe hydrochloride or oxalic acid salts for the in vitro σ₁ and σ²receptor binding assays.

EXAMPLE 2

This example illustrates synthetic steps for generating ¹⁸F-taggedcompounds of the present teachings. In this example, compounds 3c-f wereradiolabeled with ¹⁸F as shown in Schemes II-IV (FIGS. 2, 3 and 4,respectively). Scheme II (FIG. 2) outlines the synthesis of the mesylateprecursors required for the radiolabeling procedure. Alkylation of theortho hydroxyl group of compounds, 2c-e, with 1-bromoethyl acetatefollowed by hydrolysis of the acetate group produced the corresponding2-hydroxyethyl analogs, 4c-e, in good yield. Compounds, 4c-e, were thenconverted to the corresponding mesylates, 5c-e, by treatment withmethanesulfonyl chloride in dichloromethane using triethylamine as anacid scavenger.

EXAMPLE 3

This example illustrates synthesis of the precursor for thecorresponding 5-iodo analog, 5f, as shown in Scheme III (FIG. 3).Esterification of 5-bromo-2-methoxy salicylic acid followed byalkylation of the ortho hydroxyl group with 1-bromoethyl acetate, thenhydrolysis of the acetate and benzoate esters produced the corresponding2-hydroxyethyl analog, 9. Condensation of 9 with the amine, 1b, gave theamide, 4f, which was converted to the corresponding mesylate, 5f, usingthe conditions described above for the analogs, 5c-e.

EXAMPLE 4

This example illustrates synthesis of [¹⁸F]3c, [¹⁸F]3d, [¹⁸F]3e, and[¹⁸F]3f from mesylate precursors. As shown in Scheme IV (FIG. 4),mesylate precursors, compounds 5c-f, were treated with[¹⁸F]fluoride/potassium carbonate and4,7,13,16,21,24-Hexaoxa-1,10-diazabicyclo[8.8.8]-hexacosane (Kryptofix222®, Acros Organics N.V., Fairlawn, N.J.) using dimethyl sulfoxide(DMSO) as the solvent. The reaction mixture \vas irradiated for 30-40seconds in a microwave oven, and the crude product separated from theunreacted [¹⁸F]fluoride using a C-18 reverse phase Sep-Pak® cartridge(Waters Corp., Milford, Mass.) and methanol as the thaw. The crudeproduct was then purified by high-performance liquid chromatography(HPLC) using a C-18 reverse phase column. The entire procedure required˜2 h, and the radiochemical yield, corrected for decay to the start ofsynthesis, was 20-30%. The specific activities ranged from 1500-2500Ci/mmol.

EXAMPLE 5

This example illustrates in vitro binding studies with the compounds ofthe present teachings. In this example, in vitro binding studies wereconducted in order to measure the affinity of the target compounds forσ₁ and σ₂ receptors.

In these assays, the novel sigma ligands were dissolved inN,N-dimethylformamide (DMF), DMSO or ethanol, and then diluted in 50 mMTris-HCl buffer containing 150 mM NaCl and 100 mM EDTA at pH 7.4 priorto performing the σ₁ and σ₂ receptor binding assays The procedures forisolating the membrane homogenates and performing the σ₁ and σ₂ receptorbinding assays have been described in detail previously (Xu, J., et al.,Eur. J. Pharmacol. 21: 525 (1-3): 8-17, 2005. Briefly, the σ₁ receptorbinding assays were conducted in 96-well plates using guinea pig brainmembrane homogenates (˜300 μg protein) and ˜5 nM [³H](+)-pentazocine(34.9 Ci/mmol, Perkin Elmer, Boston, Mass.). The total incubation timewas 90 min at room temperature. Nonspecific binding was determined fromsamples that contained 10 μM of cold haloperidol. After 90 min, thereaction was terminated by the addition of 150 μL of ice-cold washbuffer (10 mM Tris-HCl, 150 mM NaCl, pH 7.4) using a 96 channel transferpipette (Fisher Scientific, Pittsburgh, Pa.). The samples were harvestedand filtered rapidly through a 96-well fiber glass filter plate(Millipore, Billerica, Mass.) that had been presoaked with 100 μL of 50mM Tris-HCl buffer at pH 8.0 for 1 h. Each filter was washed 3 timeswith 200 μL of ice-cold wash buffer, and the filter counted in a Wallac1450 MicroBeta liquid scintillation counter (Perkin Elmer, Boston,Mass.).

The σ₂ receptor binding assays were conducted using rat liver membranehomogenates (˜300 μg protein) and ˜5 nM [³H]DTG (58.1 Ci/mmol, PerkinElmer, Boston, Mass.) in the presence of 1 μM (+)-pentazocine to blockσ_(□) sites. The incubation time was 120 min at room temperature.Nonspecific binding was determined from samples that contained 10 μM ofcold haloperidol. All other procedures were identical to those describedfor the σ₁ receptor binding assay above.

Data from the competitive inhibition experiments were modeled usingnonlinear regression analysis to determine the concentration thatinhibits 50% of the specific binding of the radioligand (IC₅₀ value).Competitive curves were best fit to a one-site fit and gave pseudo-Hillcoefficients of 0.6-1.0. K_(i) values were calculated using the methodof Cheng and Prusoff (Biochem. Pharmacol. 22: 3099-3108, 1973) and arepresented as the mean±1 SEM. For these calculations, we used a K_(d)value of 7.89 nM for [³H](+)-pentazocine and guinea pig brain; for[³H]DTG and rat liver, we used 30.73 nM²⁰

The binding assays used [³H](+)-pentazocine for the σ₁ receptors and[³H]1,3-Di(2-tolyl)guanidine ([³H]DTG) in the presence of 100 nM(+)-pentazocine for the σ₂ receptors. The K_(i) values were determinefrom Scatchard plots. The results of the binding assays for compounds,3a-f, are shown in Table I. Increasing the length of the spacer groupfrom two carbons (3a) to 4 carbons (3c) results in a 69-fold increase inthe affinity for σ₁ receptors, a 15-fold increase in the affinity for σ₂receptors, and a 0.5 unit increase in the log D value; a measure of thelipophilicity of the compounds (Table I). Although four of the fivecompounds (3c-f) with a four-carbon spacer had higher affinities for σ₁receptors (their K_(i) values ranged from 330 to 2,150 nM) than thecompound (3a) with a two-carbon spacer (K_(i)=22,750 nM), the affinitiesof 3c-f for σ₂ receptors increased proportionately more (their K_(i)values ranged from 0.26 to 6.95 nM), leading to substantial increases intheir σ₂:σ₁ ratios (Table 1).

The σ₂:σ₁ ratios for compounds, 3c-f, varied from 48 to 8, 190. Theexcellent σ₂ receptor affinities and moderate to high σ₂:σ₁ ratios forthe compounds, 3c-f, indicated that their corresponding ¹⁸F-labeledanalogs would be useful radiotracers for imaging the σ₂ receptor statusof solid tumors with PET. Also, the log D values for these compounds, ameasure of their lipophilicity, are within the range that should lead toa high uptake in solid tumors (Xu, J., et al., Eur. J. Pharmacol. 21:525 (1-3): 8-17, 2005).

TABLE I Affinity (K_(i)) of the benzamide analogs, 6a-e, for the σ₁ andσ₂ receptors assayed in vitro K_(i) value (nM) Log D^(a) σ₁ σ₂ σ₁:σ₂Ratio (pH = 7.4) 3a 22,750 ± 3,410 102 ± 4  222 2.54 3b 15,300 ± 2,305386 ± 93  40 3.56 3c 330 ± 25 6.95 ± 1.63 48 3.06 3d 1,076 ± 88   0.65 ±0.22 1,656 3.89 3e 1,300 ± 225  1.06 ± 0.30 1,230 4.13 3f 2,150 ± 410 0.26 ± 0.07 8,190 3.46 ^(a)calculated using the program ACD/log D

EXAMPLE 6

This example illustrates in vivo evaluation of compounds of the presentteachings. All animal experiments were conducted in compliance with theGuidelines for the Care and Use of Research Animals established byWashington University's Animal Studies Committee. EMT-6 mouse mammaryadenocarcinoma cells (5×10⁵ cells in 100 uL of phosphate-bufferedsaline) were implanted subcutaneously in the scapular region of femaleBalb/c mice (˜2-month old and 17-22 g; Charles River Laboratories). Thebiodistribution studies were initiated 7-10 clays after implantationwhen the tumor size was ˜0.2 cm³ (−200 mg).

For the biodistribution studies, 10-120 μCi of [¹⁸F]3c, [¹⁸F]3d, [¹⁸F]3eor [¹⁸F]3f in 100-150 uL of saline was injected via the tail vein intoEMT-6 tumor-bearing female Balb/c mice. Groups of at least 4 mice wereused for each time point. At 5, 30, 60, and 120 min after injection, themice were euthanized, and samples of blood, lung, liver, kidney, muscle,fat, heart, brain, bone and tumor were removed, weighed and counted in aBeckman Gamma 8000 well counter. After counting, the percentage of theinjected close per gram of tissue (% ID/g) was calculated. Thetumor/organ ratios were calculated by dividing the % ID/u of the tumorby the % ID/g of each organ.

The results of the biodistribution studies in female Balb/c mice bearingEMT-6 tumors are shown in Table II. All four labeled compounds displayedexcellent tumor uptake at 5 min post-injection, with values ranging from2.5-3.7 percent of the injected dose per gram (% ID/g). Tumor uptake at1 h post-injection remained high for each of the ligands, [¹⁸F]3c,[¹⁸F]3d, [¹⁸F]3e and [¹⁸F]3f, (1.14, 2.09, 2.72, and 2.15% ID/u,respectively), and continued to remain relatively high at 2 hpost-injection (0.64, 0.96, 1.92 and 1.15% ID/g, respectively) comparedto that of the normal tissues, fat and muscle. This resulted inacceptable tumor:normal tissue ratios for the PET imaging studies. Forexample, the tumor:muscle ratios ranged from 3-4 and the tumor:fatratios ranged from 4.5-8 at 2 hrs post-injection, respectively. Also,the low bone uptake of all four labeled compounds, which continued todecrease between the 30 min and 1 h time points, suggests that thesecompounds do not undergo a significant defluorination in vivo.

Compound [¹⁸F]3f had the highest tumor:muscle ratio (˜8) and a tumor:fatratio of ˜7 at 2 h after i.v. injection (FIG. 6 A, B). The tumor:fatratios for [¹⁸F]3c and [¹⁸F]3d were also high, reaching ˜8 and ˜6,respectively, at 2 h after i.v. injection. However, the tumor: muscleratios for [¹⁸F]3c and [¹⁸F]3d were much lower than that for [¹⁸F]3f.Although the tumor uptake of [¹⁸F]3d and [¹⁸F]3e is higher than that of[¹⁸F]3c at both 1 h and 2 h post-injection, these radiotracers clearedmuch more slowly from the blood than [¹⁸F]3c (Table II), making themless desirable than [¹⁸F]3c as PET imaging agents. The moderate to hightumor: normal tissue ratios and the rapid clearance from the blood for[¹⁸F]3c and [¹⁸F]3f suggests that these radiotracers are likely the bestcandidates for imaging of solid tumors with PET. Consequently, these tworadiotracers were selected for further studies to evaluate thesuitability for detecting solid tumors and imaging their σ₂ receptorstatus with PET.

TABLE II [¹⁸F]3c-f Biodistribution in female Balb/c mice bearing EMT-6tumors 5 min. 30 min. 60 min. 120 min. 5 min. 30 min. 60 min. 120 min.[¹⁸F]3c [¹⁸F]3d blood 2.49 ± 0.49 1.16 ± 0.10 0.56 ± 0.08 0.35 ± 0.053.57 ± 0.43 2.81 ± 0.32 1.69 ± 0.63 0.52 ± 0.10 lung 10.26 ± 0.71  2.36± 0.19 0.88 ± 0.12 0.43 ± 0.07 12.08 ± 1.98  3.08 ± 0.23 1.60 ± 0.270.51 ± 0.08 liver 23.33 ± 4.22  10.51 ± 0.87  4.14 ± 0.55 2.05 ± 0.4332.60 ± 3.96  13.12 ± 1.39  5.69 ± 0.54 2.30 ± 0.33 kidney 29.18 ± 1.92 6.86 ± 0.45 2.51 ± 0.51 0.87 ± 0.13 42.94 ± 3.34  17.55 ± 2.75  6.92 ±1.61 1.12 ± 0.16 muscle 1.86 ± 0.08 0.70 ± 0.19 0.34 ± 0.05 0.24 ± 0.081.95 ± 0.19 0.98 ± 0.18 0.58 ± 0.11 0.28 ± 0.10 fat 1.95 ± 0.33 0.59 ±0.13 0.22 ± 0.04 0.08 ± 0.02 2.85 ± 0.47 0.96 ± 0.13 0.38 ± 0.05 0.15 ±0.06 heart 3.73 ± 0.15 1.15 ± 0.06 0.55 ± 0.07 0.27 ± 0.04 3.74 ± 0.371.55 ± 0.11 1.04 ± 0.21 0.40 ± 0.07 brain 0.76 ± 0.06 0.27 ± 0.05 0.18 ±0.03 0.12 ± 0.02 1.09 ± 0.11 0.40 ± 0.03 0.32 ± 0.05 0.20 ± 0.03 bone2.49 ± 0.19 0.96 ± 0.15 0.55 ± 0.07 0.45 ± 0.11 2.90 ± 0.39 1.17 ± 0.061.12 ± 0.16 1.28 ± 0.28 tumor 3.67 ± 0.45 2.54 ± 0.27 1.14 ± 0.10 0.64 ±0.10 3.28 ± 0.41 2.59 ± 0.19 2.09 ± 0.28 0.96 ± 0.24 [¹⁸F]3e [¹⁸F]3fblood 4.60 ± 0.44 4.30 ± 0.59 3.39 ± 0.29 1.92 ± 0.59 1.82 ± 0.25 1.23 ±0.28 0.65 ± 0.09 0.28 ± 0.01 lung 9.71 ± 0.83 4.07 ± 0.46 2.34 ± 0.121.36 ± 0.24 18.47 ± 3.07  3.75 ± 0.58 1.51 ± 0.13 0.74 ± 0.03 liver37.26 ± 4.88  17.35 ± 2.72  7.31 ± 0.98 4.25 ± 1.56 15.21 ± 2.21  10.73± 2.98  5.57 ± 0.31 2.61 ± 0.69 kidney 36.07 ± 2.28  17.43 ± 1.95  9.36± 0.90 3.92 ± 0.98 19.98 ± 1.66  7.73 ± 2.25 3.50 ± 0.80 1.34 ± 0.10muscle 1.52 ± 0.10 1.12 ± 0.05 0.83 ± 0.04 0.60 ± 0.11 2.50 ± 0.33 0.73± 0.14 0.40 ± 0.07 0.15 ± 0.01 fat 2.32 ± 0.46 1.04 ± 0.06 0.62 ± 0.100.44 ± 0.08 4.13 ± 0.86 1.36 ± 0.50 0.47 ± 0.07 0.17 ± 0.05 heart 3.22 ±0.27 2.37 ± 0.29 1.61 ± 0.11 1.00 ± 0.23 5.60 ± 0.45 1.54 ± 0.31 0.69 ±0.06 0.39 ± 0.04 brain 0.55 ± 0.04 0.44 ± 0.04 0.36 ± 0.02 0.37 ± 0.060.71 ± 0.09 0.27 ± 0.04 0.14 ± 0.02 0.08 ± 0.01 bone 2.59 ± 0.28 1.31 ±0.14 0.99 ± 0.07 1.67 ± 0.27 2.23 ± 0.56 2.01 ± 0.53 0.93 ± 0.16 0.59 ±0.09 tumor 2.54 ± 0.62 2.81 ± 0.62 2.72 ± 0.13 1.92 ± 0.10 3.05 ± 0.433.11 ± 0.16 2.15 ± 0.25 1.15 ± 0.23

EXAMPLE 7

This example illustrates specificity of binding in vivo for a, receptorsby compounds of the present teachings.

In order to demonstrate that the in vivo binding of [¹⁸F]3c and [¹⁸F]3fis specific for σ₂ receptors, a no-carrier-added dose of theseradiotracers was co-injected into EMT-6 tumor-bearing mice withN-(4-fluorobenzyl)piperidinyl-4-(3-bromophenyl)acetamide (YUN-143), asigma ligand displaying a high affinity for both σ₁ and σ₂ receptors.Co-injection of YUN-143 with either [¹⁸F]3c or [¹⁸F]3f resulted in asignificant decrease (˜50%) in the tumor:muscle and tumor:fat ratios at1 h post-injection (FIG. 7).

These blocking studies in tumor-hearing mice were conducted byco-injecting 1 mg/kg of coldN-(4-fluorobenzyl)piperidinyl-4-(3-bromophenyl)acetamide (YUN-143) with[¹⁸F]3c or [¹⁸F]3f. Yun-143 has a high affinity for both σ₁ and σ₂receptors and is routinely used for sigma receptor blocking studies(Mach, R. H., et al., Nucl Med. Biol. 28: 451-458, 2001; Bowen, W. D. etal., Eur. J. Pharmacol. 278: 257-260, 1995; Bonhaus, D. W. et al., J.Pharmacol. Exp. Ther. 267: 961, 1993. All mice were sacrificed 60 minafter injection of the radiotracer, and the tumor:organ ratios weredetermined as described above. The data presented in FIG. 7 indicatethat both [¹⁸F]3c and [¹⁸F]3f bind selectively to σ₂ receptors in vivo.

EXAMPLE 8

This example illustrates use of radioligands of the present teachings asimaging agents.

To confirm the feasibility of using radioligands of the presentteachings as PET imaging agents for determining the σ₂ receptor statusof solid tumors, a CT/PET study using either [¹⁸F]3c or [¹⁸F]3f infemale Balb/c mice bearing EMT-6 tumors was performed on a microPET-F220(CTI-Concorde Microsystems Inc.) and a MicroCAT-II system (ImTek Inc.).For the microPET studies, each mouse was injected with ˜0.25 mCi ofeither [¹⁸F]3c or [¹⁸F]3f via the tail vein and imaged 1 h later.MicroCT images were also obtained and co-registered with the PET imagesto determine the exact anatomical location of the radiotracers.

In these studies, the EMT-6 tumors were readily identifiable usingeither radioligand, indicating that they are both acceptable agents fordetecting solid tumors and imaging their σ₂ receptor status with PET(FIG. 8, EMT-6 Mouse Mammary Tumors).

EXAMPLE 9

This example describes a general method for synthesis of the substituted2-hydroxybenzoic acid amides, compounds 2a-e, in particular compound 2a.

To synthesizeN-[2-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-ethyl]-2-hydroxy-5-methyl-benzamide(compound 2a), 1,3-dicyclohexycarbodimide (432.6 mg, 2.1 mmol) and1-hydroxybenzotriazole (283.8 mg, 2.10 mmol) were added to a ice-waterbath cooled solution of 1a (472.0 mg, 2.0 mmol) and2-hydroxy-5-methyl-benzoic acid (152 mg, 2.0 mmol) in 30 mldichloromethane. After the reaction mixture was stirred overnight,analysis of the products using thin layer chromatography with 20%methanol and 80% ethyl ether as the mobile phase indicated that thereaction was complete. Alter completion of the reaction, another 50 mlof dichloromethane was added to the mixture. The organic solution wasthen washed with an aqueous saturated NaHCO₃ solution and brine,sequentially. The organic solution was dried with anhydrous sodiumsulfate. After removal of the solvent, the crude product was purified bycolumn chromatography using 20% methanol and 80% ethyl ether as themobile phase. The yield of 2a was 37.1%. The ¹H-NMR spectrum (300 MHz,CDCl₃) of the purified product was: 2.25 (s, 3H), 2.75-2.95 (m, 6H),3.58-3.65 (m, 4H), 3.82-3.83 (s, 6H), 6.48-6.51 (s, 1H), 6.62-6.63 (s,1H), 6.82-6.85 (d, 1H), 7.02 (s, 1H), 7.08 (s, 1H), 7.20 (d, 1H). LCMSm/e: 371.2 (M+H).

EXAMPLE 10

This example illustrates synthesis of5-Bromo-N-[4-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-butyl]-2-hydroxy-3-methoxy-benzamide(compound 2b).

Compound 2b was prepared from 5-bromo-2-hydroxy-3-methoxy-benzoic acidand 1b as described above for 2a. The yield of 21) was 16.7%. The ¹H-NMRspectrum (300 MHz, CDCl₃) of the purified product was: 1.73-1.76 (m,4H), 2.57-2.59 (m, 2H), 2.76-2.81 (m, 4H), 3.45-3.47 (m, 2H), 3.58-3.61(m, 2H), 3.82 (s, 3H), 3.86 (s, 3H), 3.88 (s, 3H), 6.48-6.51 (t, 1H),6.56-6.59 (t, 1H), 6.97-7.00 (m, 1H), 7.07-7.10 (m, 1H). LCMS m/e:493.10 (M+H).

EXAMPLE 11

This example illustrates synthesis ofN-[4-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-butyl]-2-hydroxy-5-methyl-benzamide(compound 2c).

Compound 2c was prepared from 2-hydroxy-5-methyl-benzoic acid and 1b asdescribed above for 2a. The yield of 2c was 45%. The ¹H-NMR spectrum(300 MHz, CDCl₃) of the purified product was: 1.75 (m, 4H), 2.12 (s,3H), 2.58 (m, 2171), 2.75-2.77 (m, 2H), 2.82-2.84 (m, 2H), 3.40-3.50 (m,2H), 3.58 (s, 2H), 3.83 (s, 3H), 3.85 (s, 3H), 6.50 (s, 1H), 6.60 (s,1H), 6.85-6.88 (d, 1H), 7.06 (s, 1H), 7.13-7.16 (d, 2H), 7.61 (s, 1H).LCMS m/e: 399.20 (M+H).

EXAMPLE 12

This example illustrates synthesis ofN-[4-(6,7-dimethoxy-3,4-dihydro-1H-isbquinolin-2-yl)-butyl]-2-hydroxy-5-bromo-benzamide(compound 2d).

Compound 2d was prepared form 5-bromo-2-hydroxy-benzoic acid and 1b asdescribed above for 2a. The yield of 2d was 28.0%. The ¹H-NMR spectrum(300 MHz, CDCl₃) of the purified product was: 1.71-1.81 (m, 4H),2.55-2.85 (m, 6H), 3.44-3.48 (m, 2H), 3.58-3.60 (m, 2H), 3.82 (s, 3H),3.89 (s, 3H), 6.50 (s, 1H), 6.59 (s, 1H), 6.82-6.84 (d, 1H), 7.30-7.40(d, 1H), 7.52 (d, 1H), 8.30 (s, 1H). Anal. (C₂₂H₂₇BrN₂O₄.1.25H₂O) C, H,N.

EXAMPLE 13

This example illustrates synthesis ofN-[4-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-butyl]-2-hydroxy-5-iodo-benzamide(2e).

Compound 2e was prepared form 2-hydroxy-5-iodo-benzoic acid and 1b asdescribed above for 2a. The yield of 2c was 27.0%. The ¹H-NMR spectrum(300 MHz, CDCl₃) of the purified product was: 1.69-1.81 (m, 4H),2.54-2.65 (m, 2H), 2.75-2.83 (m, 2H), 3.44-3.48 (m, 2H), 3.58 (s, 2H),3.82 (s, 3H), 3.85 (s, 3H), 6.50 (s, 1H), 6.58 (s, 1H), 6.70-6.74 (d,1H), 7.54-7.55 (d, 1H), 7.65-7.67 (d, 1H), 8.20 (s, 1H). Anal.(C₂₂H₂₇IN₂O₄.0.75H₂O) C, H, N.

EXAMPLE 14

This example describes a general method for synthesis of the substituted2-(2-fluoroethoxy)benzoic acid amides, 3a-e, in particular compound 3a.

To synthesize[N-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-ethyl]-2-(2-fluoro-ethoxy)-5-methyl-benzamide(compound 3a), potassium carbonate (792.5 Mg, 4.88 mmol) was added to asolution of 2a (278 mg, 0.75 mmol) and 2-bromo-1-fluoroethane (620 mg,4.88 mmol) in acetone (60 mL). The reaction mixture was refluxed for 48h until the reaction was complete as determined by thin layerchromatography with 5% methanol and 95% ethyl ether as the mobile phase.The solvent was evaporated, 30 ml of water added to the flask, and thenthe mixture extracted with dichloromethane (25 mL×3). After the organiclayer was dried with anhydrous sodium sulfate, the crude product waspurified by column chromatography using 5% methanol and 95% ethyl etheras the mobile phase. The yield of 3a was 90%. The ¹H-NMR spectrum (300MHz, CDCl₃) of the purified product was: 2.33 (s, 3H), 2.64-2.80 (m,6H), 3.63-3.75 (m, 4H), 3.84 (s, 3H), 3.86 (s, 3H), 4.16 (m, 1H), 4.21(m, 1H), 4.41 (m, 1H), 4.60 (m, 1H), 6.55 (s, 1H), 6.61 (s, 1H),6.78-6.81 (d, 1H), 7.20 (d, 1H), 8.00 (s, 1H), 8.28 (s, 1H). LCMS m/e:417.22 (M+H). For the in vitro binding experiments, the free base wasconverted into the hydrochloride salt; m.p. 159-161° C., Anal.(C₂₃H₃₀ClFN₂O₄). C. H. N.

EXAMPLE 15

This example illustrates synthesis of5-bromo-N-[4-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-butyl]-2-(2-fluoro-ethoxy)-3-methoxy-benzamide(compound 3b).

Compound 3b was prepared from 2b as described for 3a above. The yieldwas 50%. The ¹H-NMR spectrum (300 MHz, CDCl₃) of the purified productwas: 1.64 (m, 4H), 2.48-2.52 (t, 3H), 2.64-2.66 (t, 3H), 2.74-2.78 (t,3:H), 3.42-3.55 (m, 4H), 3.79-3.87 (m, 9H), 4.19-4.22 (t, 2H), 4.29-4.32(t, 2H), 4.58-4.61 (t, 2H), 4.74-4.77 (t, 2H), 6.47 (s, 1H), 6.54 (s,1H), 7.07 (d, 1H), 7.78 (d, 1H), 8.10 (s, 1H). For the in vitro bindingexperiments, the free base was converted into the oxalic acid salt; m.p.127-129° C. LCMS m/e: 590.30 (M+Li). Anal. (C₂₆H₃₃BrFN₂O₇).

EXAMPLE 16

This example illustrates synthesis ofN-[6,7-dimethoxy-3,4-clihydro-1H-isoquinolin-2-yl-butyl]-2-(2-fluoro-ethoxy)-5-methyl-benzamide(compound 3c).

Compound 3c was prepared from 2c as described above for 3a. The yield of3c was 67%. The ¹H-NMR spectrum (300 MHz, CDCl₃) of the purified productwas: 1.67-2.00 (m, 4H), 2.33 (s, 3H), 2.51-2.56 (t, 3H), 2.67-2.72 (t,3H), 3H), 2.78-2.82 (t, 3H), 3.48-3.54 (m, 4H), 3.82 (s, 3H), 3.83 (s,3H), 4.21-4.24 (t, 1H), 4.30-4.33 (t, 1H), 4.68-4.71 (t, 1H), 4.84-4.87(t, 1H), 6.49 (s, 1H), 6.57 (s, 1H), 6.79-6.82 (d, 1H), 7.20 (m, 1H),7.96 (s, 1H), 7.99-7.80 (d, 1H). LCMS m/e: 445.25 (M+H). For the invitro binding experiments, the free base was converted into the oxalicacid salt; m.p. 131-133° C. Anal. (C₂₆H₃₄FN₂O₆).

EXAMPLE 17

This example illustrates synthesis ofN-[4-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-butyl]-2-(2-fluoro-ethoxy)-5-bromo-benzamide(compound 3d).

Compound 3d was prepared from 2d as described above for 3a. The yield of3d was 38.90%. The ¹H-NMR spectrum (300 MHz, CDCl₃) of the purifiedproduct was: 1.57-1.80 (m, 4H), 2.62-2.66 (m, 3H), 2.78-2.82 (m, 3H),3.48-3.51 (m, 2H), 3.60-3.64 (m, 2H), 3.82 (s, 3H), 3.83 (s, 3H),4.21-4.25 (t, 1H), 4.31-4.35 (t, 1H), 4.70-4.74 (t, 1H), 4.86-4.90 (t,1H), 6.49 (s, 1H), 6.57 (s, 1H), 6.78-6.82 (d, 1H), 7.48-7.52 (d, 1H),7.96 (s, 1H), 8.26 (d, 1H). LCMS m/e: 509.1 (M+H). For the in vitrobinding experiments, the free base was converted into the oxalic acidsalt; m.p. 119-121° C. Anal. (C₂₅H₃₁BrEN₂O₆).

EXAMPLE 18

This example illustrates synthesis ofN-[4-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-butyl]-2-(2-fluoro-ethoxy)-5-iodo-benzamide(compound 3e).

Compound 3e was prepared from 2e as described above for 3a. The yield of3e was 41.4%. The ¹H-NMR spectrum (300 MHz, CDCl₃) of the purifiedproduct was: 1.61-1.72 (m, 4H), 2.46-2.52 (m, 2H), 2.67-2.71 (m, 2H),2.76-2.78 (m, 2H), 3.41-3.47 (m, 2H), 3.52 (t, 2H), 3.80 (s, 3H), 3.83(s, 3H), 4.19-4.21 (m, 1H), 4.27-4.31 (m, 1H), 4.67-4.71 (m, 1H),4.83-4.86 (m, 1H), 6.46 (s, 1H), 6.55 (s, 1H), 6.62-6.66 (d, 1H),7.62-7.67 (m, 1H), 7.87 (s, 1H), 8.40-8.41 (d, 1H). LCMS m/e: 557.13(M+H). For the in vitro binding experiments, the free base was convertedinto the oxalic acid salt; imp. 121-123° C. Anal. (C₂₅H₃₁FIN₂O₆).

EXAMPLE 19

This example describes a general method for synthesis of the substituted5-bromo-benzoic acid derivatives into their substituted5-tributylstannanyl benzoic acid derivatives in particular compound 3g.

To synthesizeN-[4-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-butyl]-2-(2-fluoro-ethoxy)-3-methoxy-5-tributylstannanyl-benzamide(compound 3g), Nitrogen was bubbled for 5-10 min through a solution of3b (200 mg, 0.371 mmol) in 20 ml fresh distilled toluene. The wholesystem was covered with aluminum foil. Tetrakis(triphenylphosphinepalladium(0) [(PPh₃)₄Pd(0)] (42 mg, 0.036 mmol) and bis(tributylin)[Sn(C₄H₉)₃]₂ (575 mg, 0.99 mmol) was added to the reaction mixture andheated overnight at 110° C. with an oil bath. Thin layer chromatographywith 45% hexane, 45% ethyl ether and 10% methanol as the mobile phasewas used to assess when the reaction was complete. After quenching thereaction, the crude product was purified on a silica gel column toisolate the tin intermediate, 3g. The yield of 3g was 64%. The ¹H-NMRspectrum (300 MHz, CDCl₃) of the purified product was: 0.87-1.58 (m,27H), 1.61-1.69 (m, 4H), 2.56 (s, 2H), 2.72 (s, 2H), 2.81-2.82 (s, 2H),3.47-3.49 (d, 2H), 3.57 (s, 2H), 3.83 (s, 6H), 3.88 (s, 3H), 4.25 (d,1H), 4.37 (s, 1H), 4.62-4.65 (s, 1H), 4.78-4.81 (s, 1H), 6.51 (s, 1H),6.58 (s, 1H), 7.81 (s, 1H), 8.07 (s, 1H). LCMS m/e: 747.60 (M−H).

EXAMPLE 20

This example illustrates a general method for converting the tinprecursor of the benzoic acid derivatives into their correspondingiodine substituted benzoic acid derivatives, in particular compound 3f.

To synthesize(N-[4-(6,7-Dimethoxy-3,4-dihydro-1.1H-isoquinolin-2-yl)-butyl]-2-(2-fluoro-ethoxy)-3-methoxy-5-iodo-benzamide(31), a solution of iodine in CHCl₃ (5 mL, 0.5 M) was added dropwise toa solution of the tin precursor, 3g (258 mg, 0.34 mmol) in 20 ml CH₂Cl,until the color of the solution persisted. The reaction was stirred atroom temperature for 30 min, and a solution of 5% aqueous NaHSO₃ wasadded until the solution was colorless. The mixture was extracted withCH₂Cl₂, and the organic layers washed with brine before being dried withNa₂SO₄. The organic layers were then concentrated under vacuum andpurified using a silica gel column with 15% methanol and 85% ether asthe mobile phase to isolate 3f. The yield of 3f was 36%. The ¹H-NMRspectrum (300 MHz, CDCl₃) of the purified product was: 1.60-1.80 (m,2H), 1.80-2.10 (m, 4H), 3.19-3.2 (m, 2H), 3.40-3.50 (m, 2H), 3.68 (m,2H), 3.83 (m, 2H), 3.92 (s, 3H), 3.95 (s, 3H), 3.99 (s, 3H), 4.26 (t,1H), 4.37 (s, 1H), 4.65 (s, 1H), 4.80 (s, 1H), 6.50 (s, 1H), 6.58 (s,1H), 7.28 (d 1H), 8.02 (d, 1H), 8.21 (s, 1H). LCMS m/e: 587.14 (M+H).For the in vitro binding experiments, the free base was converted intothe oxalic acid salt; m.p. 125-127° C. Anal. (C₂₆H₃₃FIN₂O₇).

EXAMPLE 21

This example describes a general method for converting the substituted2-hydroxy benzoic acid derivatives into their substituted2-(2-hydroxy-ethoxy)-benzoic acid amides, in particular compound 4c.

To synthesizeN-[4-6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-butyl]-2-(2-hydroxy-ethoxy)-5-methyl-benzamide(compound 4c), anhydrous potassium carbonate (546.0 mg, 3.26 mmol) wasadded to a solution of 2c (200.0 mg, 0.5 mmol) and 2-bromoethyl acetate(547.0 mg, 3.27 mmol) in 60 mL of acetone. The reaction mixture wasrefluxed for 48 h under nitrogen. After 48 h, thin layer chromatographywith 15% methanol and 8.5% ether as the mobile phase indicated that thereaction was complete. After evaporating the solvent, the residue wasdissolved in 30 ml of water and extracted with ethyl acetate (20×3 mL).Then the organic component was washed with brine, dried with anhydroussodium sulfate, concentrated, and the final product purified on a silicagel column to isolate the2-{2-[4-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-butylcarbamoyl]-4-methyl-phenoxy}-ethylester. The yield of this intermediate was 82.2%. The ¹H-NMR spectrum(300 MHz, CDCl₃) of the purified product was: 1.70 (m, 4H), 2.01 (s,3H), 2.33 (s, 3H), 2.56 (m, 2H), 2.71-2.73 (m, 2H), 2.81 (m, 2H),3.50-3.52 (m, 2H), 3.55 (s, 2H), 3.83 (s, 3H), 3.84 (s, 3H), 4.23 (t,2H), 4.50 (t, 2H), 6.50 (s, 1H), 6.58 (s, 1H), 6.78-6.82 (d, 1H),7.18-7.25 (d, 1H), 7.95 (s, 1H), 8.02 (s, 1H).

NaOH (30 mg, 0.75 mmol) was added to a solution of this intermediate(182 mg, 0.375 mmol) in 20 mL of methanol and 10 mL of water. Thereaction mixture was stirred overnight until the reaction was complete.Then 0.375 mL of 2 N HCl was added to neutralize the solution. Afterevaporating the solvent, the residue was dissolved in 60 mL of ethylacetate. The solution was washed first with water, then brine, andfinally dried with anhydrous sodium sulfate. After evaporating thesolvent, the crude product was purified on a silica gel column. Theyield of 4c was 96%. The ¹H-NMR spectrum (300 MHz, CDCl₃) of thepurified product was: 1.68-1.85 (m, 4H), 2.39 (s, 3H), 2.45 (s, 1H),2.51-2.61 (m, 2H), 2.80-2.87 (m, 4H), 3.45-3.61 (m, 4H), 3.76-3.80 (t,2H), 3.83 (s, 3H), 3.85 (s, 3H), 3.83 (s, 3H), 4.05-4.08 (t, 2H), 6.49(s, 1H), 6.60 (s, 1H), 6.79-6.83 (d, 1H), 7.15-7.19 (d, 2H), 7.93 (s,1H), 8.30 (s, 1H).

EXAMPLE 22

This example illustrates synthesis of5-Bromo-N-[4-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-butyl]-2-(2-hydroxy-ethoxy)-benzamide(compound 4d).

Compound 4d was prepared from 2d as described above for 4c. The yield ofcompound 4d was 70%. The ¹H-NMR spectrum (300 MHz, CDCl₃) of thepurified product was: 1.65-1.90 (m, 4H), 2.66-2.70 (m, 2H), 2.92 (m,4H), 3.51-3.54 (m, 2H), 3.72 (m, 2H), 3.72-3.81 (t, 2H), 3.83 (s, 3H),3.85 (s, 3H), 4.05-4.09 (t, 2H), 6.51 (s, 1H), 6.60 (s, 1H), 6.77-6.81(d, 1H), 7.44-7.48 (d, 2H), 8.17 (s, 1H), 8.30 (s, 1H).

EXAMPLE 23

This example illustrates synthesis ofN-[4-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-butyl]-2-(2-hydroxy-ethoxy)-5-iodo-benzamide(compound 4e).

Compound 4e was prepared from 2e as described above for 4c. The yield of4e was 77%. The ¹H-NMR spectrum (300 MHz, CDCl₃) of the purified productwas: 1.60-1.90 (m, 4H), 2.63-2.66 (m, 3H), 2.90 (s, 2H), 3.52-3.55 (m,2H), 3.70 (m, 2H), 3.79-3.82 (t, 2H), 3.83 (s, 3H), 3.85 (s, 3H),4.08-4.10 (t, 2H), 6.50 (s, 1H), 6.60 (s, 1H), 6.67-6.70 (d, 1H),7.60-7.70 (d, 1H), 8.30 (s, 1H), 8.40-8.41 (d, 1H).

EXAMPLE 24

This example describes a general method for converting the substituted2-hydroxy-ethoxy benzoic acid amides of the present teachings to theirmethanesulfonic acid esters, in particular compound 5c.

To synthesize2-(2-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butylcarbamoyl)-4-methylphenoxy)ethylmethanesulfonate (compound 5c), methanesulfonic chloride (120 mg, 1.04mmol) was added to an ice-water cooled solution of 4c (354 mg, 0.8 mmol)and triethylamine (242 mg, 2.4 mmol) in 30 mL of dichloromethane. Thereaction mixture was stirred for 3 h until thin layer chromatographyusing 5% methanol and 95% dichloromethane as the mobile phase indicatedthat the reaction was complete. After 3 h, 20 mL of dichloromethane wasadded, the solution was washed with first a saturated sodium carbonateaqueous solution (20 mL×3), then brine, and finally dried with anhydroussodium sulfate. After evaporating the solvent, the crude product waspurified on a silica gel column to isolate 5c. The yield of 5c was81.6%. The ¹H-NMR spectrum (300 MHz, CDCl₃) of the purified product was:2.07-2.10 (m, 4H), 2.71 (s, 3H), 2.80-3.00 (m, 2H), 3.08-3.20 (m, 4H),3.40 (m, 3H), 3.80-4.00 (m, 4H), 4.20 (s, 3H), 4.22 (s, 3H), 4.60-4.68(t, 2H), 4.95-4.97 (t, 2H), 6.88 (s, 1H), 6.95 (s, 1H), 7.15-7.18 (d,1H), 7.50-7.60 (d, 1H), 8.20 (s, 1H), 8.19 (s, 1H). Anal. (C₂₆H₃₆N₂O₇S)C, H, N.

EXAMPLE 25

This example illustrates synthesis of2-(4-bromo-2-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butylcarbamoyl)phenoxy)ethylmethanesulfonate (compound 5d).

Compound 5d was prepared from 4d as described above for 5c. The yield of5d was 77%. The ¹H-NMR spectrum (300 MHz, CDCl₃) of the purified productwas: 1.72-1.75 (m, 4H), 2.61-2.68 (m, 2H), 2.85 (s, 3H), 3.05 (s, 2H),3.42-3.58 (m, 4H), 3.68 (s, 2H), 3.82 (s, 3H), 3.83 (s, 3H), 4.29 (t,2H), 4.60 (t, 2H), 6.50 (s, 1H), 6.57 (s, 1H), 6.70-6.77 (d, 1H),7.45-7.55 (d, 1H), 7.95 (s, 1H), 8.20-8.22 (d, 1H). LCMS m/e: 585.10(M+H).

EXAMPLE 26

This example illustrates synthesis of2-(2-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butylcarbamoyl)-4-iodophenoxy)ethylmethanesulfonate (compound 5e).

Compound 5e was prepared from 4c as described above for 5c. The yield of5e was 80%. The ¹H-NMR spectrum (300 MHz, CDCl₃) of the purified productwas: 1.60-1.80 (m, 4H), 2.52-2.55 (On, 2H), 2.67-2.70 (m, 2H), 2.70-2.78(m, 2H), 3.02 (s, 3H), 3.46-3.52 (m, 4H), 3.82 (s, 3H), 3.83 (s, 3H),4.23-4.26 (t, 2H), 4.56-4.60 (t, 2H), 6.48 (s, 1H), 6.55 (s, 1H),6.60-6.64 (d, 1H), 7.64-7.68 (d, 1H), 7.85 (s, 1H), 8.38-8.39 (d, 1H).LCMS m/e: 633.10 (M+H).

EXAMPLE 27

This example illustrates synthesis of2-(2-Acetoxy-ethoxy)-5-bromo-3-methoxy-benzoic acid methyl ester(compound 7).

To prepare this compound, initially 1.0 mL of 98% concentrated sulfuricacid was added to a solution of 5-bromo-2-hydroxy-3-methoxy-benzoicacid, (compound 6) (1.0 g, 4.0 mmol) in 50 of methanol. The reactionmixture was refluxed overnight until thin layer chromatography using 20%ethyl acetate and 80% hexane as the mobile phase indicated that thereaction was complete. After evaporating the methanol, the residue wasdissolved in 60 mL of ethyl acetate and washed with a saturated NaHCO₃aqueous solution and then brine. After drying with anhydrous sodiumsulfate, the solution was concentrated, and the crude product waspurified on a silica gel column to isolate the intermediate,5-bromo-2-hydroxy-3-methoxy-benzoic acid methyl ester. The yield of thisintermediate was 94%. The ¹H-NMR spectrum (300 MHz, CDCl₃) of thepurified product was: 3.88 (s 3H), 3.95 (s, 3H), 7.09 (d, 1H), 7.55 (t,1H), 10.96 (d, 1H).

Potassium carbonate (2.90 g, 21.0 mmol) was added to a solution of theabove intermediate (0.84 g, 3.23 mmol) and 2-bromoethyl acetate (3.5 g,20.96 mmol) in 60 mL of acetone. The reaction mixture was refluxed for72 h until thin layer chromatography using 20% ethyl acetate and 80%hexane as the mobile phase indicated that the reaction was complete.After evaporating the solvent, the residue was dissolved in 30 mL ofwater and then extracted with ethyl acetate (25 mL×3). The organicsolution was dried with anhydrous sodium sulfate, resuspended, andpurified on a silica gel column. The yield of 7 was 78%. The ¹H-NMRspectrum (300 MHz, CDCl₃) of the purified product was: 2.10 (s, 3H),3.86 (s, 3H), 3.88 (s, 3H), 4.21-4.24 (t, 2H), 4.38-4.41 (t, 3H), 7.14(s, 1H), 7.45 (s, 1H).

EXAMPLE 28

This example illustrates synthesis of2-(2-Acetoxy-ethoxy)-5-iodo-3-methoxy-benzoic acid methyl ester(compound 8).

To synthesize compound 8, nitrogen was bubbled for 5-10 min through asolution of 2-(2-acetoxy-ethoxy)-5-bromo-3-methoxy-benzoic acid methylester, (compound 7) (270 mg, 0.778 mmol) in 20 mL of freshly distilledtoluene. The reaction system was covered with aluminum foil.Tetrakis(triphenylphosphine) palladium(0) [(PPh₃)₄Pd(0)] (100 mg, 0.087mmol) and bis(tributlytin) [Sn(C₄H₉)₃]₂ (899 mg, 1.55 mmol) were addedto the reaction mixture and heated overnight at 110° C. in an oil-bathwhile stirring. After quenching, thin layer chromatography using 15%ethyl acetate and 85% hexane as the mobile phase indicated that thereaction was complete. The product was then purified on a silica gelcolumn to isolate the tin precursor,2-(2-acetoxy-ethoxy)-3-methoxy-5-tributylstannanyl-benzoic acid methylester. The yield of the tin precursor was 37.3%. The H-NMR spectrum (300MHz, CDCl₃) of the purified product was: 0.8-1.75 (m, 27H), 2.10 (s,3H), 3.87 (s, 3H), 3.89 (s, 3:H), 4.24-4.27 (t, 2H), 4.38-4.41 (t, 2H),7.09-7.30 (s 1H), 7.35 (s 1H).

A solution of iodine in CHCl₃ (5 mL, 0.5 M) was added dropwise to asolution of the above tin precursor (680 mg, 1.22 mmol) in 20 mL ofCH₂Cl₂ until the color of the solution persisted. Then the reaction wasstirred at room temperature for 30 min, and a quench solution of 5%aqueous NaHSO₃ was added until the solution became colorless. Themixture was extracted with CH₂Cl₂, and the organic layers washed withbrine and dried by Na₂SO₄. The organic layers were then condensed undervacuum and purified using a silica gel column with 15% ethyl acetate and85% hexane as the mobile phase. The yield of compound 8 was 90%. The¹H-NMR spectrum (300 MHz, CDCl₃) of the purified product was: 2.10 (s,3H), 3.85 (s, 3H), 3.88 (s, 3H), 4.21-4.25 (t, 2H), 4.37-4.41 (t, 2H),7.29-7.30 (s, 1H), 7.63 (s, 1H).

EXAMPLE 29

This example illustrates synthesis of2-(2-Hydroxy-ethoxy)-5-iodo-3-methoxy-benzoic acid (compound 9)

Compound 9 was prepared from compound 8 as described in the generalmethod for converting the substituted 2-hydroxy benzoic acid derivativesinto their substituted 2-(2-hydroxy-ethoxy)-benzoic acid amides (Example21). The yield of compound 9 was 81%. The ¹H-NMR spectrum (300 MHz,CDCl₃) of the purified product was: 3.89 (s, 3H), 3.93-3.96 (t, 2H),4.33-4.36 (t, 2H), 7.08 (s, 1H), 7.62 (s, 1H).

EXAMPLE 30

This example illustrates synthesis ofN-[4-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-butyl]-2-(2-hydroxy-ethoxy)-5-iodo-3-methoxy-benzamide(compound 4f)

Compound 4f was prepared from 9 and 1.1) as described in the generalmethod for synthesis of the substituted 2-hydroxybenzoic acid amides(Example 9). The yield of 4f was 29%. The ¹H-NMR spectrum (300 MHz,CDCl₃) of the purified product was: 1.72-1.75 (m, 4H), 2.56 (m, 2H),2.75-2.77 (m, 2H), 2.81-2.83 (m, 2H), 3.49-3.51 (m, 2H), 3.55 (s, 2H),3.56-3.60 (t, 2H), 3.82 (s, 3H), 3.83 (s, 3H), 3.85 (s, 3H), 4.06-4.10(t, 2H), 6.47 (s, 1H), 6.57 (s, 1H), 6.90 (s, 1H), 7.57 (s, 1H),7.70-7.80 (s, 1H).

EXAMPLE 31

This example illustrates synthesis of2-(2-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butylcarbamoyl)-4-iodo-6-methoxyphenoxy)ethylmethanesulfonate (compound 50.

Compound 5f was prepared from 4f as described in the general method forconverting the substituted 2-hydroxy-ethoxy benzoic acid amides of thepresent teachings to their methanesulfonic acid esters (Example 24). Theyield of 5f was 61%. The ¹H-NMR spectrum (300 MHz, CDCl₃) of thepurified product was: 1.72 (m, 4H), 2.55 (s, 2H), 2.70 (d, 2H), 22.76(d, 2H), 3.05 (s, 3H), 3.85 (m, 9H), 4.26 (m, 2H), 4.49 (m, 2H), 6.49(s, 1H), 6.55 (s, 1H), 6.93 (d, 1H), 7.51 (m, 1H), 8.02 (s, 1H). LCMSm/e: 663.20 (M+H). Anal. (C₂₅H₃₂FIN₂O₅) C: Calcd, 51.20. found 36.61; C:Calcd, 5.50. found 4.34; N: Calcd, 4.78. found 3.12.

EXAMPLE 32

This example illustrates production of [¹⁸F]Fluoride.

[¹⁸F]Fluoride was produced in our institution by proton irradiation ofenriched ¹⁸O water (95%) [reaction: ¹⁸O(p, n)¹⁸F] using either a JSWBC-16/8 (Japan Steel Works) or a CS-15 cyclotron (Cyclotron Corp).

EXAMPLE 33

This example illustrates a general method for labeling the substituted2-(2-fluoroethoxy)benzoic acid amide analogs with ¹⁸F, in particular[¹⁸F](N-[4-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-butyl]-2-(2-fluoro-ethoxy)-3-methoxy-5-iodo-benzamide(compound [¹⁸F]3f).

For this synthesis, [¹⁸F]fluoride (100-150 mCi) was added to a 10-mLPyrex screw cap tube containing 5-6 mg of Kryptofix 222 and 0.75 ma ofK₂CO₃. Using HPLC grade acetonitrile (3×1.0 mL), the water wasazeotropically evaporated from this mixture at 110° C. under a stream ofargon. After all of the water was removed, a solution of the precursor,5f, (1.5-2.0 mg) in DMSO (0.2 mL) was added to the reaction vesselcontaining the ¹⁸F/Kryptofix mixture. A 3 mm glass bead was added to thereaction vessel to insure a more homogeneous heat distribution when thesample was irradiated with microwaves, and the vessel capped firmly on aspecially designed remotely operated capping station. After vortexing,the reaction mixture was irradiated with microwaves for 30-40 sec atmedium power (60 Watts) until the thin layer chromatography scanner witha 25% of methanol and 75% dichloromethane mobile phase indicated thatthe incorporation yield was 40-60%.

After adding 6 mL of water and shaking, the solution was loaded on aC-18 reverse phase Waters Oasis cartridge (H LB-6 cc) that hadpreviously been rinsed with a solution of 5% methanol in water (5-8 mL).The sample was then rinsed 3 times with 6 mL water to eliminate theunreacted fluoride. The retained activity was eluted with 5-8 mL ofacetonitrile. After evaporating the acetonitrile to a volume of <0.5 mL,the sample was loaded on a C-18 Alltech econosil semi-preparative HPLCcolumn (250×10 um). The product was eluted with 29% acetonitrile and 71%0.1 M ammonium formate buffer at a flow rate of 4.5 mL/min. Theretention time of the [¹⁸F]3f was ˜33 min. The solution containing the[¹⁸F]3f was concentrated, resuspended in saline, and a 100 uL aliquotsent for quality control analysis before using it in the biodistributionand imaging studies. The entire procedure required ˜2 h.

Quality control analysis was performed on an analytical EPLC system thatconsisted of an Alltech econosil reversed phase C-18 column (250×4.6 mm)with a mobile phase of 35% acetonitrile and 65% 0.1 M ammonium formatebuffer at pH 4.0-4.5. At a flow rate of 1.2 mL/min, the [¹⁸f]3f elutedat 13.2 min with a radiochemical purity of >99%. The labeling yield was˜30% (decay corrected), and the specific activity was >2000 Ci/mmol.

EXAMPLE 34

This example illustrates synthesis of[¹⁸F](N-[4-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-butyl]-2-(2-fluoro-ethoxy)-5-methyl-benzamide(compound [¹⁸H]3c).

Compound [¹⁸F]3c was prepared from 5c as described above for [¹⁸F]3fwith the following exceptions. The semi-preparative HPLC mobile phasewas 39% methanol and 61% 0.1 M formate buffer. At a flow rate of 3.5mL/min, the [¹⁸F]3c eluted at ˜33 min with a radiochemical purityof >99%. The labeling yield was ˜35% (decay corrected), and the specificactivity was >1500 Ci/mmol. The entire procedure took ˜2 h.

To check that the chemical characteristics of [¹⁸F]3c were identical tothe cold standard, 3c, both compounds were run on the analytical HPLCsystem with a mobile phase of 52% methanol and 48% 0.1 M formate buffer.At a flow rate of 1.5 mL/min, the two compounds co-eluted with aretention time of 4.7 min.

EXAMPLE 35

This example illustrates synthesis of[¹⁸F](N-[4-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-butyl]-2-(2-fluoro-ethoxy)-5-bromo-benzamide(compound [¹⁸F]3d).

Compound [¹⁸F]3d was prepared from 5d as described above for [¹⁸F]3fwith the following exceptions. The semi-preparative HPLC mobile phasewas 13% of THF and 87% 0.1 M formate buffer. At a flow rate of 3.5mL/min, the [¹⁸F]3d eluted at ˜20 min with a radiochemical purityof >98%. The labeling yield was ˜30% (decay corrected), and the specificactivity was >1500 Ci/mmol. The entire procedure took ˜2 h.

To check that the chemical characteristics of [¹⁸F]3d were identical tothe cold standard, 3d, both compounds were run on the analytical HPLCsystem with a mobile phase of 38% acetonitile and 62% 0.1 M formatebuffer. At a flow rate of 1.5 mL/min, the two compounds co-eluted with aretention time of ˜8 min.

EXAMPLE 36

This example illustrates synthesis of[¹⁸F](N-[4-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-butyl]-2-(2-fluoro-ethoxy)-5-Iodo-benzamide(compound [¹⁸F]3c).

Compound [¹⁸F]3e was prepared from 5c as described above for [¹⁸F]3fwith the following exceptions. The semi-preparative HPLC mobile phasewas 15% THF and 85% 0.1 M formate buffer. At a flow rate of 6.0 mL/min,the [¹⁸F]3e eluted at ˜35 min with a radiochemical purity of >99%. Thelabeling yield was ˜30% (decay corrected), and the specific activitywas >1500 Ci/mmol. The entire procedure took ˜2 h.

To check that the chemical characteristics of [¹⁸F]3e were identical tothe cold standard, 3e, both compounds were run on the analytical HPLCsystem with a mobile phase of 13% acetonitrile and 87% 0.1 M formatebuffer. At a flow rate of 2.0 mL/min, the two compounds co-eluted with aretention time of 15.2 min.

EXAMPLE 37

This example provides an elemental analysis of various compounds of thepresent teachings. The data are presented in Table V.

TABLE V Elemental Analysis Calculated Measured Compounds Formula C H N CH N 2d C₂₂H₂₇BrN₂O₄•1.25H₂O 54.38 6.12 5.77 54.44 5.74 5.69 2eC₂₂H₂₇IN₂O₄•0.75H₂O 50.44 5.48 5.35 50.45 5.27 5.24 5c C₂₆H₃₆N₂O₇S 59.986.97 5.38 59.89 6.94 5.49 3a C₂₃H₃₀ClFN₂O₄•H₂O 58.66 6.85 5.95 58.946.65 6.01 3b C₂₆H₃₃BrFN₂O₇•0.5H₂O 52.62 5.77 4.72 53.29 6.27 3.98 3cC₂₆H₃₄FN₂O₆•H₂O 61.52 7.15 5.52 61.84 6.95 5.27 3d C₂₅H₃₁BrFN₂O₆•1.5H₂O51.64 5.89 4.82 51.32 5.47 4.59 3e C₂₅H₃₁FIN₂O₆•1.75H₂O 47.44 5.49 4.4347.07 5.09 4.11

EXAMPLE 38

This example illustrates, in FIG. 9 (Tumor Imaging Study: σ₂ vs. FDG),imaging of a glioma using [¹⁸F]3f of the present teachings compared to[¹⁸F]FDG. Note greater contrast using compound [¹⁸F]3f as a radiotracer.

Patents and publications discussed herein are hereby incorporated byreference.

1. A method for synthesizing a radiolabelled fluoroalkoxybenzamide compound of structure

the method comprising: forming a mixture comprising i) an organic solvent, ii) a compound of structure

iii) ¹⁸F/4,7,13,16,21,24-Hexaoxa-1,10-diazabicyclo[8.8.8]-hexacosane/K₂CO₃; and heating the mixture, wherein m is an integer from 1 to about 10, n is an integer from 1 to about 10, R₁ and R₂ are each independently selected from the group consisting of H, a halogen selected from the group consisting of Br, Cl and F, a C₁₋₄ alkoxy, a C₁₋₄ alkyl, a C₁₋₄ fluoroalkyl, a C₁₋₄ fluoroalkoxy, CF₃, OCF₃, SCH₃, SCF₃, and NH₂.
 2. A method for synthesizing a radiolabelled fluoroalkoxybenzamide compound in accordance with claim 1, wherein m=2, n=4, R₁ is selected from the group consisting of H and OCH₃, and R₂ is selected from the group consisting of CH₃, Br and I.
 3. A method for synthesizing a radiolabelled fluoroalkoxybenzamide compound in accordance with claim 1, wherein m=2, n=4, R₁ is OCH₃, and R₂ is I.
 4. A method for synthesizing a radiolabelled fluoroalkoxybenzamide compound in accordance with claim 1, wherein the organic solvent is dimethyl sulfoxide.
 5. A method for synthesizing a radiolabelled fluoroalkoxybenzamide compound in accordance with claim 1, wherein the organic solvent is acetonitrile. 